Creatinine iminohydrolase is an enzyme which specifically hydrolyzes creatinine to ammonia. Accordingly, by contacting an aqueous liquid containing creatinine with this enzyme to generate ammonia, the presence and/or concentration of creatinine in the liquid can be determined by detecting the level of generated ammonia. This enzyme can therefore play an important role in the clinical laboratory where it can be used as a diagnostic test reagent for the determination of creatinine in biological liquids.
Creatinine iminohydrolase, sometimes referred to as creatinine desimidase, has been obtained from various microorganisms. For example, J. Szulmajster in J. Bacteriolol, 75: 633 (1958) and in Biochim Biophys Acta, 30: 154 (1958) describes a preparation of creatinine iminohydrolase obtained from the anaerobic, gram-positive microorganism Clostridium paraputrificum. A method of growing the Clostridium paraputrificum microorganism is also described in these Szulmajster publications. However, these publications relate specifically to an anaerobic, gram-positive microorganism, and the disclosed fermentation method for growing the microorganism requires a long time. Moreover, the amount of microbial cells grown and the yield of enzyme extracted therefrom is relatively small. Accordingly, as of the filing date of the present specification, large-scale production of enzyme from the microbial source Clostridium paraputrificum does not appear to be practical.
U.S. Pat. Nos. 4,087,329 and 4,134,793 describe the production of the enzyme creatinine desimidase from one of several aerobic microbial sources including microorganisms of the genera Brevibacterium, Corynebacterium, Pseudomonas, and Arthrobacter. These patents further describe a nutrient medium which may be used for culturing microorganisms of the aforementioned genera. These patents assert that the formulation of this nutrient medium can be widely varied and can contain any of a large number of specifically recited carbon and nitrogen sources, as well as other optional nutrients including inorganic materials, a creatinine inducer, and the like.
Goodhue, Esders, and Masurekar copending U.S. Patent Application Ser. No. 091,218, filed concurrently herewith and entitled "Creatinine Iminohydrolase Free From Urease Activity" describes and claims a new creatinine iminohydrolase enzyme preparation free from urease activity obtained from an aerobic soil microorganism, preferably the aerobic soil microorganism ATCC 31546. Because this enzyme preparation is free of urease contamination and is highly specific for creatinine, creatinine assays can be performed with this enzyme without regard to interference by urea and other nitrogenous substances that are often present in biological aqueous liquids to be assayed for creatinine, e.g., serum. The new urease-free creatinine iminohydrolase enzyme preparation described by Goodhue, Esders, and Masurekar in the aforementioned patent application is therefore highly desirable.
An improved process and aqueous nutrient medium for growing a creatinine iminohydrolase-producing aerobic soil microorganism and increasing the yield of the creatinine iminohydrolase from the microorganism would represent a clearly advantageous addition to the art. Such a process and medium would be particularly desirable if useful with the aerobic soil microorganism ATCC 31546 noted above.
McCollough, Esders and Lynn, U.S. Patent Application Ser. No. 091,217 filed concurrently herewith entitled "Process For The Recovery Of Intracellular Enzymes" describes and claims an improved method for the extraction of intracellular enzymes such as urease-free creatinine iminohydrolase from an aerobic soil microorganism. The foregoing Goodhue, Esders and Masurekar and McCollough, Esders and Lynn copending patent pulications are incorporated by reference herein.